EP0135378A1

EP0135378A1 - Reagent for quantitative determination of microorganisms

Info

Publication number: EP0135378A1
Application number: EP84305727A
Authority: EP
Inventors: Tsunehiro Kitagawa
Original assignee: Dainippon Pharmaceutical Co Ltd
Current assignee: Sumitomo Pharma Co Ltd
Priority date: 1983-08-24
Filing date: 1984-08-22
Publication date: 1985-03-27
Also published as: JPH0510627B2; DE3469226D1; US4693968A; JPS6046461A; EP0135378B1

Abstract

An agent for quantitative determination of microorganisms comprises

(a) an antibody prepared from a strain of the same species as the species of microorganisms to be determined,
(b) an insolubilized cell component of a strain which can sufficiently bind to the antibody (a) but does not release and replace other strains which have been bound with the antibody (a), and
(c) a labelled second antibody.

Quantitative determination of the microorganisms can be carried out by (i) adding a predetermined amount of component (a) to a test sample, which component (a) is present in excess, to cause an antigen - antibody reaction, (ii) adding component (b) for reaction thereof with the unreacted antibody, (iii) separating the precipitated antigen - antibody complex, (iv) adding component (c), (v) washing the mixture and (vi) measuring the activity of labelled substance in the complex. The agent and method of the present invention are useful for determining simultaneously not only a strain from which the antibody is prepared but also other strains of the same species which are contained in the test sample.

Description

The present invention relates to an agent for quantitative determination of microorganisms. More particularly, it relates to an agent for quantitative determination of microorganisms comprising the following components:

(a) an antibody prepared from a strain of the same species as the species of microorganisms to be determined,
(b) an insolubilized cell component of a strain which can sufficiently bind to the antibody (a) but does not release and replace to other strains which have been bound with the antibody (a), and
(c) a labelled second antibody.

Various microorganisms such as bacteria, actinomycetes, fungi cause rot of foods and various diseases in animals and plants, and hence, it is very important to determine quantity of the microorganisms in order to know the degree of rot or degree of infection. There have hitherto been known various methods for the quantitative determination of microorganisms, for example, (i) a method of counting the number of colonies of microorgnisms by naked eyes, and (ii) a method of determining impedance, change of pH value, or amount of consumed oxygen, produced carbon dioxide, metabolites, or produced enzymes, which are induced by the presence of microorganisms. However, these methods (i) and (ii) must be applied to the test sample after pre-treatment for inhibiting growth of other microorganisms, and hence, they require much time for preparation thereof. Besides, the method must be done after culturing for 24 hours in order to grow the microorganism to be determined, because, otherwise, the sensitivity is very low. Thus, these methods can not be applied to rapidly.
It is recently tried to determine the microorganisms by utilizing antigen-antibody reaction. For example, Sarafian et al. have tried to determine an antigen common to all strains of genococci by sandwiching whole cells, lipopolysaccharides or outer membrane proteins of the genococcus with an anti-genococcus whole cell rabbit antibody and an insolubilized anti-genococcus whole cell mouse antibody, followed by adding an enzyme-labelled anti-rabbit IgG goat antiserum [cf. J. Med. Microbiol., 15, 541 - 550 (1982)]. The outer membrane proteins are an antigen common to all strains, and the outer membrane prcteins obtained from each strain are determined in a single system in the above method. However, in order to apply this method to the quantitative determination of microorganisms, the microorganisms must be cultured in order to extract the outer membrane proteins therefrom, which is troublesome, and further, such a common antigen is not necessarily present in all microorganisms other than genococci. Besides, Dodd et al. have tried to determine Escherichia coli by competing the test sample microorganism with irsclubilized flagella of E. coli against an antibody prepared from the fla^gella of E. coli, followed by adding thereto an enzyme-labelled second antibody [cf. Infect. Immun., 38, 764 - 773 (1962)]. According to this method, the collected test samples can be used as they stand with high sensitivity. Although the strain used for the preparation of an antibody can be determined with very high sensitivity by this method, other strains can almost not be determined. For example, in case of determining E. coli contained in foods, it is impossible to determine total number of all of E. coli strains contained in the foods.
Thus, according to the conventional methods for the quantitative determination of microorganisms, only a specified strain can be determined among the microorganisms contained in the test sample, but total or some groups of strains of the microorganisms can not simultaneously be determined.
The present inventor has studied on an improved method for the quantitative determination of microorganisms without the drawbacks in the known methods, and has thought that the reason why the known methods can determine merely a specific strain is that the strain to be used for the preparation of an antibody for insolubilization (or the strain for insolubilizing) is the same as the strain to be used for the preparation of the antibody. Then, the present inventor has used an insolubilized strain which can sufficiently bind with the antibody but the binding is weak and has tried to apply said strain to the test sample in the conventional competition method. As a result, it has been found that the insolubilized strain has a low affinity to the antibody and the amount of antibody bound to the insolubilized strain is very low, and hence, such a method can not practically be used.
As a result of a further study, it has now been found that the desired quantitative determination of microorganisms can be done by subjecting an excess amount of an antibody prepared from a strain of the same species as the species of the microorganisms to be determined to an antigen-antibody reaction with the microorganisms contained in the test sample, and then counting the remaining antibody which has not reacted with the microorganisms in the test sample. That is, it has been found that when an insolubilized cell component of a strain which can sufficiently bind to the free antibody but does not release and replace to other strains which have been bound with the antibody is used and the remaining antibody which has not reacted with the microorganisms in the test sample is bound with the insolubilized strain, the antibody bound with the insclubilized cell component is easily quantitatively determined, from which the quantity of the antibody bound to the strains in the test sample (in turn the quantity of the strains in the test sample) can be calculated backwards.
An object of the present invention is to provide an improved agent for the quantitative determination of microorganisms, which can be useful for simultaneous determination of a-group of strains of the same species in the microorganisms contained in the test sample. Another object of the invention is to provide a method for the quantitative determination of a group of strains of species in the microorganisms contained in test samples. These and other objects and advantages of the present invention will be apparent to those skilled in the art from the following description.
The agent for quantitative determination of microorganisms of the present invention comprises the following components:

According to the present invention, the determin- aticn is carried out by the steps of (i) adding an excess and predetermined amount of the antibody (a) to the test sample in order to subject them to an antigen-antibody reaction, (ii) adding the insolubilized cell component (b) to the reaction mixture in order to react the remaining antibody which has not reacted with the antigen : (microorganisms) contained in the test sample, (iii) separating the precipitated antibody (a) - antigen (b) complex, (iv) adding thereto the labelled second antibody (c) , (v) washing the mixture with phosphate buffer, and then measuring the activity of labelled substance in the complex. According to the above method, the amount of the antibody in foe solid phase is determined, and hence, the amount of the antibody in the liquid phase is calculated, based on which the amount of microorganisms contained in the test sample can be quantitatively determined.
The agent of the present invention is usually in the form of a kit, and may optionally be used in a combination with a standard solution containing a predetermined amount of microorganism useful for preparing a calibration curve, an agent for determining the activity of the labelled substance (e.g., in case of enzyme-labelled antibody, the agent comprises a substrate, a substrate- diluent, an enzyme-reaction terminater, etc.), and a buffer, in addition to the above components (a) to (c).
The antibody (a) is usual prepared by disrupting the culture cells of a strain of microorganisms to separate the cell components (e.g. cell walls, etc.), mixing the separated components with an appropriate adjuvant, administering subcutaneously or intramuscularly the mixture to animals such as rabbit, guinea pi^g, gcat, sheep, etc., collecting a blood serum from the animals, and treating the collected serum in a usual manner. The strain may be any strain among the same species of the microorganisms which are contained in the test sample.
The insolubilized cell component (b) can be prepared by culturing a strain which can sufficiently bind tc the antibody (a) but does not release the binding of the antibody (a) with other strains (antigen) contained in the test sample, disrupting the culture cells to separate the cell components (e.g. cell walls, etc.), and binding the separated cell components to an insoluble carrier, such as natural insoluble polysaccharides, chemically treated dextrane gels, agar gels, plastic beads, acrylamide gels, glass beads, metal oxide powders, synthetic rubber tube, or the like. When the cells per se are insolubilized, they may disadvantageously bind with other microorganisms. For the binding, various binding agents are used depending on the insoluble carriers, for example, glutalaldehyde, toluenediisocyanate, dihalogeno-nitrobenzene, etc. for chemically binding the amino group in the cell walls with the amino group in the insoluble carrier, and maleimide derivatives [e.g. N-((-maleimidobutyloxy)succinimide, etc.] or the like for binding the amino group in the cell walls with the thiol group in the insoluble carrier.
The labelled second antibody (c) can be prepared by labelling an antibody (second antibody) against γ-globulin (IgG) of the animal which is used for the preparation of the antibody (a). The labelling is carried out by using enzymes, radioisotopes, fluorescent compounds, spin compounds, etc. In case of an enzyme-labelled second antibody, it is easily prepared by binding an enzyme with the second antibody in the same manner as in the binding between the cell walls and the insoluble carrier as mentioned above. Suitable examples of the enzyme are β-galactosidase, peroxidase, lipase, alkaliphosphatase, glucose-6-phosphate dehydrogenase, etc.
The agent of the present invention is advantageous in that the test sample can be immediately applied to without culturing and can be useful for comprehensively determining a group of strains of species in the microorganisms contained in the test sample, and is particularly useful for the quantitative determination of bacteria (e.g. Escherichia coli, Streptococcus mutans), actinomycetes (e.g. Streptomyces scabies), fungi (e.g. Pvricularia oryzae), or the like.
Embodiments of the present invention are illustrated by the following Examples.

Example 1

Preparation of antiserum of Streptomyces scabies Obama strain

(i) Preparation of cell walls:

Streptomyces scabies Obama strain is inoculated in a medium (200 ml) containing K₂HPO₄ (0.1 g), asparagin (0.1 g), glucose (2 g) and 15 % extract of potato (40 ml), and the mixture is cultured with shaking for 7 days. The cells are collected by filtration, washed with purified water twice and then lyophilized to give dried cells (1 g). The dried cells thus obtained are suspended in a 0.02 M phosphate buffer containing 0.9 % sodium chloride (100 ml), and the mixture is treated with a ultrasonic disintegrator (Branson Sonifier W 185 type, 60 W) for 15 minutes under ice-cooling. The resulting mixture is centrifuged at 2,200 rpm for 10 minutes, and the precipitates are collected, washed twice with purified water (5 ml) and then lyophilized to give the desired cell walls (0.1 g), which is kept at 4°C.

(ii) Immunization

Cell walls (0.5 mg) obtained above are suspended in 0.9 % NaCl (0.5 ml) and thereto is added an incomplete Freund's adjuvant (0.5 ml) to emulsify the mixture. The emulsion is subcutaneously or intramuscularly injected to rabbit. The injection is repeated 4 times at two weeks intervals, and ten days after the final injection, the rabbit is bled.

Example 2

Preparation of antiserum of E. coli K 12 strain:

(i) Preparation of cell walls:

E. coli K 12 strain is inoculated in a bouillon medium (manufactured by Difco Laboratories, U.S.A., 2,000 ml) and the mixture is cultured with shaking overnight. The cells are collected by centrifuging at 20,000 x g for 30 minutes, washed twice with 0.02 M phosphate buffer containing 0.15 M sodium chloride and then lyophilized to give dried cells (2 g). The dried cells thus obtained are suspended in a 0.02 M sodium chloride-containing phosphate buffer (100 ml), and the mixture is treated with a ultrasonic disintegrator (the same as used in Example 1) for 10 minutes under ice-cooling. The resulting mixture is centrifuged at 20,000 x g for 30 minutes, and the precipitates are collected, washed twice with purified water (20 ml) and then lyophilized to give the desired cell walls (0.1 g), which is kept at 4°C.

(ii) Immunization

The cell walls obtained above are treated in the same manner as described in Example 1 (ii) to give the desired antiserum.

Example 3

Preparation of antiserum of Pyricularia oryzae 001 Kyu 82-05A strain:

(i) Preparation of cell walls:

In the same manner as described in Example 1 (i) except that Pyricularia oryzae 001 Kyu 82-05A strain is used and a medium containing KH₂PO₄ (0.1 g), K₂HPO₄ (0.1 ^g), MgSO₄ (0.1 g), CaCl₂ (0.02 g), glucose (4 g) and yeast extract (1 g) is used as the medium, there are obtained the desired cell walls.

(ii) Immunization

Example 4

Preparation of antiserum of Streptococcus rutans No. 6715 strain:

(i) Preparation of cell walls:

S. mutans No. 6715 strain is inoculated in a brain heart infusion-medium (manufactured by BBL Co., 1,000 ml) and cultured. The cells are collected by centrifuging at 10,000 x g for 15 minutes, washed twice with distilled water, and lyophilized to give dried cells (0.6 g). The dried cells thus obtained are suspended in 0.02 M phosphate buffer containing 0.15 M sodium chloride (100 ml) and the mixture is treated with a ultrasonic disintegrator (the same as used in Example 1) for 15 minutes under ice-cooling. The resulting precipitates are collected by centrifuging at 20,000 x g for 30 minutes, washed twice with purified water (20 ml) and then are again treated with a ultrasonic disintegrator under the same condition as above for 45 minutes. The resulting mixture is lyophilized to give the desired cell walls (0.1 g), which is kept at 4°C.

(ii) Immunization

The cell walls obtained above (0.5 mg) are suspended in a 0.9 % Nacl (0.5 ml) and thereto is added an incomplete Freund's adjuvant (0.5 ml) to emulsify the mixture. The emulsion is intravenously injected to rabbit. The injection is repeated 3 times at two weeks intervals, and just before each injection, the rabbit is bled. The cellected blood is kept at -30°C.

Example 5

Preparation of an insolubilized antigen:

An Amino-Dylark cylinder (manufactured by Sekisui Kagaku K.K., hereinafter referred to as "AD") which is washed with a detergent, SCAT-20X (manufactured by Daiichi Kogyo Seiyaku, K.K.) is immersed in a 1 % glutaraldehyde for 1 hour and then washed with 0.01 M phosphate buffer (pH 7.0) containing 0.9 % sodium chloride. The aldehyde-introduced AD thus obtained (100 cylinders) are immersed in a suspension of cell walls (100 µg) of Streptomyces scabies Aino strain, E. coli No. 45 strain, Pyricularia oryzae 031 Ine 72 strain or Streptococcus mutans HS-6 strain [which is prepared in the same manner as described in Examples 1 to 4, (i)] in 0.02 M phosphate buffer containing 0.9 % sodium chloride (10 ml), and the mixture is shaken at room temperature for 0.5 hour and at 4°C for 2 hours. The reaction mixture is washed twice with a 0.01 M EDTA - 0.1 % BSA-containing 0.06 M phosphate buffer (pH 7.4) (hereinafter, referred to as "Buffer A") to give the desired insclubilized antigen, which is kept in Buffer A at 4°C.

Example 6

Preparation of an enzyme-labelled second antibody: To an anti-rabbit IgG goat serum (manufactured by Miles corporation, U.S.A., 5 ml) is added a 0.1 M phosphate buffer (pH 7.0, 5 ml), and to the mixture is added an aqueous saturated ammonium sulfate (10 ml) under ice-cooling, and the mixture is stirred for 20 minutes and then centrifuged at 12,000 x g for 10 minutes to collect the precipitates. The above procedure is repeated twice. The precipitates thus obtained are dissolved in a 0.1 M phosphate buffer (pH 7.5, 5 ml). The solution is dialyzed against a 0.9 % NaCl-0.02 M phosphate buffer (pH 7.0) (2,000 ml) at 4°C for 24 hours to give an anti-rabbit IgG goat serum IgG fraction. The fraction (6 ml) thus obtained is dissolved in a 0.1 M phosphate buffer (pH 6.0) (1 ml), and the solution is mixed with a solution (0.5 ml) of N-
maleimidobutyloxy)succinimide (1 mg/ml) in tetrahycrofuran.
The mixture is stirred at room temperature for 30 minutes. The reaction mixture (50 µl) is added to a solution of β-galactosidase (500 µg) in a 0.1 M phosphate buffer (pH 6.0) (1 ml), and the mixture is stirred for 30 minutes. The reaction mixture is passed through a column of Sepharose 6B (manufactured by Pharmacia, Sweden) (2 cmφ x 38 cm) which is equilibrated with a 0.1 M NaCl - 1 mM MgCl₂ - 0.1 % BSA - 0.1 % NaN₃ containing 0.02 M phosphate buffer (pH 7.0) (hereinafter, referred to as "Buffer B"), and then eluted with Buffer B, wherein each fraction contains 3 ml of eluate. The enzymatic activity of each fraction is measured by the method of Aikawa, T. et al [cf. Endocrinology, 105, 1-6 (1979)], and a fraction having a peak enzymatic activity is collected to give the desired β-galactosidaze-labelled second antibody.

Example 7

Qantitative determination of a test sample containing microorganisms:

To a test sample containing Pyricularia orvzae (100 µl) is added a 20,000 folds diluted solution (100µl) of the antiserum prepared in Example 3 in a test tube, and the mixture is reacted at room temperature overnight. To the reaction mixture is added-the insolubilized antigen prepared in Example 5 in an excess amount, and the mixture is shaken at 25°C for one hour. The reaction mixture is washed twice with Buffer B (1 ml), and thereto is added the β-galactosidase-labelled second antibody (0.2 ml) prepared in Example 6 (the β-galactosidase activity: 200 µu; 1 U means an amount necessary for hydrolyzing 1 µmol of the substrate for 1 minute), and the mixture is shaken at 30°C for 4 hours. The mixture is washed twice with Buffer B (1 ml), and thereto is added 0.1 mM 7-β-D-galactopyranosyloxy-4-methylcoumarine (150 µl), and the mixture is incubated at 33°C for 30 minutes. To the reaction mixture is added a 0.2 M glycine-NaOH buffer (pH 10.3, a reaction terminater) (2.5 ml) to terminate the enzyme reaction. The fluorescent intensity of 7-hydroxy-4-methylcoumarine produced by the enzyme reaction is measured spectrofluorometerically.

A standard curve is drawn on a semilogarithmic coordinate paper, as shown in the accompanying Figure 1, wherein the ordinate axis is relative bound enzyme activity (B/Bo %) and the abscissa axis is a concentration of P^yricularia oryzae (ng/tube). According to this method, 1 ng to 100 ng of Pyricularia oryzae can be measured per one test tube.
In the same manner as described above, the content cf E. coli, Streptomyces scabies and Streptococcus mutans can be measured.

Example 8

Comparison of cross reactivity due to the difference of the strains of microorganisms used for solid phase:

In the same manner as described in Exanple 7 except that an antibody prepared from Pyricularia oryzae 001 Kyu 82-05A strain and an insolubilized product of said strain were used, the quantitative determination of the strain was done. As a result, only the same strain as used above was determined.

Among various stains of Pyricularia oryzae as shown in the followng Table 1, 037 Ken 60-19 strain was elected, and an insolubilized product of said strain was used as the standard strain and subjected to the determination likewise. As a result, it showed a cross reactivity with other strains as shown in Table 1. Besides, when an insolubilized strain of 031 Ine 72 strain which showed the most week cross reactivity among the test strains was used and subjected to comparison of the cross reactivity, likewise. The result is shown in Table 2.
As is clear from the comparison of the data of Table 1 and Table 2, the reactivity of all strains shown in Table 2 increased and all strains other than 001 Kyu 82-05A strain can be determined. Besides, it is clear from Table 2 that any strains of other species is not determined.

Example 9

Comparison of cross reactivity due to the difference of the strains used for solid phase:

In the same manner as described in Example 7 except that an antiboty prepared from Streptococcus mutans No. 6715 strain and an insolubilized product of said strain vere used, the quantitative determination of the strain was done. As a result, the strain showed the cross reactivity as shown in the accompanying Fig. 2. The quantitative determination was done likewise by using an insolubilized product of HS-6 strain which showed the weakest cross reactivity among the strains in Fig. 2. The result is shown in Fig. 3.

As is clear from the comparison of the data in Fig. 2 and Fig. 3, each strain in Fig. 3 showed increased reactivity, which means that strains other than No. 6715 strain can also be sufficiently determined.

Example 10

Accuracy and precision of the value measured by the agent of the present invention:

A test sample containing strains of Pyricularia orvzae was repeatedly tested 5 times within a day (i.e. intra-assay) and further tested one time per day for 5 days (i.e. inter-assay), and the mean of the data and the standard deviation were calculated. The results are shown in Table 3.

As is clear from Table 3, the agent of the present invention showed less difference in intra-assay and also in inter-assay, which means that the agent of the present invention can be used for the quantitative determination of microorganisms with high accuracy and precision.

Claims

1. An agent for quantitative determination of microorganisms, which comprises

(a) an antibody prepared from a strain of the same species as the species of microorganisms to be determined,

(b) an insolubilized cell component of a strain which can sufficiently bind to the antibody (a) but does not release and replace other strains which have been bound with the antibody (a), and

(c) a labelled second antibody.

2. The agent according to claim 1, wherein the antibody (a) is an antiserum obtained by immunizing an animal with cell walls of the strain, and the insolubilized cell component (b) is cell walls of the strain which are insolubilized with a conventional insoluble carrier.

3. The agent according to claim 1 or 2, wherein the compenents (a), (b) and (c) are in the form of a kit.

4. A method for the quantitative determination of macrcorganisms, which comprises the steps of (i) adding an excess and predetermined amount of an antibody prepared from a strain of the same species as the species of microorganisms to be determined to the test sample in order to subject them to an antigen-antibody reaction, (ii) adding an insolubilized cell component of a strain which can sufficiently bind to the antibody but does not release and replace other strains which have been bound with the antibody to the reaction mixture in order to react the remaining antibody which has not been reacted with the antigen (microorganisms) contained in the test sample, (iii) separating the precipitated antibody - antigen complex, (iv) adding thereto a labelled second antibody, (v) washing the mixture, and then (vi) measuring the activity of labelled substance in the complex.